When cultured on plastic culture dishes for several days, alveolar type II cells gradually lose both their morphologic and biochemical identifying characteristics. Although type II cells cultured on a matrix derived from corneal endothelial cells have previously been reported to retain lamellar bodies for 7-10 days in culture, the ability of type II cells cultured on matrix to synthesize surfactant lipids has not been previously studied. We therefore measured the phospholipid content and the distribution of [14C]acetate into classes of lipids by type II cells maintained in culture. We found no differences between cells cultured on plastic or on matrix. We then studied the binding to type II cells in culture of Maclura pomifera and Ricinus communis I, lectins specific in vivo for type II and type I cells, respectively. We found that the cells progressively bind less M. pomifera and more R. communis I. The change in pattern of lectin binding occurs whether cells are cultured on plastic or matrix, whether lectins are conjugated with fluorescein, rhodamine or ferritin, or whether cells are cultured in the presence or absence of serum. We conclude that type II cells cultured on either tissue culture plastic or matrix derived from corneal endothelial cells lose the ability to synthesize and contain surfactant phospholipids, and, at least in their pattern of lectin binding, become similar to type I cells.